The present invention relates to a novel process for labelling a synthetic or natural ribonucleic acid (RNA).
A synthetic RNA is to be understood as meaning an RNA which is obtained by a technique which was developed by man, for example an amplification technique (PCR followed by a transcription) or a transcriptional amplification technique (TMA). A natural RNA is to be understood as meaning an RNA which is obtained by extraction from a cell, for example a messenger RNA, a ribosomal RNA or a transfer RNA.
The state of the art shows that there are a large number of methods for labelling nucleotides, oligonucleotides or nucleic acids; oligonucleotides and nucleic acids will be referred to by the term polynucleotides. Oligonucleotides can be labelled either during synthesis or by incorporating at least one labelled nucleotide.
A first method consists in attaching the label to the base, whether the latter is a natural base or a modified base. A second method proposes attaching the label to the sugar, again whether the latter be a natural sugar or a modified sugar. A third method relates to attaching the label to the phosphate.
Labelling the base was used, in particular, in the approach of labelling nucleic acids by incorporating directly labelled nucleotides.
Labelling the sugar is often used in the case of nucleic acid probes which are prepared by chemical synthesis.
Labelling the phosphate has also been used for introducing functionlized arms and labels when synthesizing the polynucleotides chemically.
In fact, the skilled person who is to label a nucleotide or a nucleotide analogue or a nucleic acid is inclined to attach the label to the base or to the sugar, which offer him more convenience and more options. This is, furthermore, what emerges from studying a large number of documents such as EP-A-0.329.198, EP-A-0.302.175, EP-A-0.097.373, EP-A-0.063.879, U.S. Pat. No. 5,449,767, U.S. Pat. No. 5,328,824, WO-A-93/16094, DE-A-3.910.151 and EP-A-0.567.841 in the case of the base or EP-A-0.286.898 in the case of the sugar.
Nevertheless, these techniques suffer from a number of drawbacks, the two main ones being the steric hindrance and the effects which are engendered by the presence of a label.
When the base is labelled, the steric hindrance is due to the encroachment of the label on the space where a neighbouring base is present, irrespective of whether this neighbouring base is carried by an adjacent nucleotide of the same strand or by the complementary strand. It is also quite obvious that the presence of the label on the base can impair efficacy and specificity during enzymic incorporation and can have an effect on the quality of the hydrogen bonds between the two complementary strands, something which can be injurious to hybridization.
When the sugar is labelled, the steric hindrance is due to the encroachment of the label on the space where an adjacent sugar carried by the same strand is present. This presence of the label can move apart two adjacent bases which are carried by the same strand and, as a consequence, prevent satisfactory hybridization with the complementary strand due to the fact that the hydrogen bonds between the strands are not optimal.
The technique of attaching the label to the phosphate is more complex than the technique involved in functionalizing the base or the sugar.
Even so, some documents have proposed techniques for labelling the phosphate. This applies, for example, to document EP-A-0.280.058, which describes labelling a nucleotide by attaching the label to the phosphate, with the latter being attached to the sugar in the 2xe2x80x2 and/or 5xe2x80x2 positions, when the nucleotide is a deoxyribonucleotide, and in the 2xe2x80x2, 3xe2x80x2 and/or 5xe2x80x2 positions when the nucleotide is a ribonucleotide. This document also describes a polynucleotide or oligonucleotide which comprises at least one labelled nucleotide as described above; this nucleotide is incorporated into the polynucleotide or oligonucleotide during synthesis.
However, the labelling which is proposed by document EP-A-0.280.058 does not enable the nucleic acids to be labelled uniformly. This is because the incorporation of the labelled nucleotides into the polynucleotides cannot be controlled; it depends entirely on the composition of polynucleotides which is to be synthesized. Thus, some polynucleotides may contain a large number of labelled nucleotides whereas others may not contain any at all. As a result, the intensity of the signal emitted by these nucleic acids will not be uniform, something which could easily make it difficult to interpret the results when detecting the nucleic acids.
In this case, the labelling is incorporated biologically without there being any control of the positions of the labelled nucleotides.
The document U.S. Pat. No. 5,317,098 relates to nucleic acids which are labelled at their 5xe2x80x2 ends. This attachment uses imidazole and a linker arm. There is no associated fragmentation. Furthermore, phosphate is added; kinase is therefore used.
Nevertheless, a phosphate will logically be present at each free end of the nucleic acid, leading to at least one additional step. This labelling is not associated with any fragmentation.
In addition, the labelling described by the preceding two documents is carried out on large nucleic acids. Thus, no fragmentation stage, also termed a cleavage stage, wets described before the labelling steps. As a result, the duplexes formed after hybridization are not stable when these target nucleic acids are hybridized to capture probes. This also applies when the polynucleotides are used as detection probes. The reasons may be due to steric hindrance or to a lack of specificity between the polynucleotide, which has been synthesized, and its target, which is not necessarily of the same size. This will therefore result in a quantitative and qualitative loss of the signal.
Steric hindrance may not only be the result of the length of the nucleic acid but also of the existence or the conservation of secondary structures. Fragmentation makes it possible to destroy these structures and in this way to optimize hybridization. This steric hindrance plays a particularly important role in the case of hybridization to surfaces which contain a high density of capture probes, for example the DNA chips developed by the company Affymetrix (xe2x80x9cAccessing Genetic Information with High-Density DNA arraysxe2x80x9d, M. Shee et al., Science, 274, 610-614. xe2x80x9cLight-generated oligonucleotide arrays for rapid DNA sequence analysisxe2x80x9d, A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 5022-5026). In this technology, the capture probes are generally of reduced size, being of about twenty nucleotides.
A large number of methods are described in the state of the art for fragmenting nucleic acids.
In the first place, the fragmentation can be enzymic, i.e. the nucleic acids can be fragmented by nucleases (DNases or RNases). This generates small fragments having 3xe2x80x2-OH, 5xe2x80x2-OH, 3xe2x80x2-phosphate and 5xe2x80x2-phosphate ends.
In the second place, the fragmentation can be chemical. For example, in the case of DNAs, it is possible to depurinate or depyrimidinate the DNAs, which are then fragmented in the presence of a base by a mechanism termed xe2x80x9cxcex2-eliminationxe2x80x9d. The DNAs can be fragmented by oxidation, alkylation or free radical addition mechanisms, inter alia. Metal cations, which are often combined with organic molecules used as chemical catalysts, for example imidazole, are used for fragmenting RNAs. This fragmentation is preferably carried out in an alkaline medium and generates fragments having 3xe2x80x2-phosphate ends.
However, the objective of these fragmentations is not that of facilitating or permitting labelling.
Document WO-A-88/04300 proposes a method for fragmenting and labelling RNA, with the fragmentation being carried out using RNA which possesses enzymic properties, i.e. ribozymes. With each cleavage, this fragmentation by ribozymes releases a nucleic acid (5xe2x80x2) HO end and a nucleic acid (3xe2x80x2) HOxe2x80x94PO2 end. The labelling, which is solely radioactive labelling, is then effected using an incorporation enzyme (kinase), which incorporates an added radioactive phosphate which is derived from a molecule of xcex3-GTP. This attachment is effected solely at the 5xe2x80x2 end. Furthermore, the fragmentation is only carried out by ribozymes, implying the existence of a specificity between the ribozymes and the target nucleic acids to be cleaved. The phosphate then acts as the label.
Our invention attaches a label to the only phosphate of a nucleic acid fragment which is released during the cleavage. There is no specificity that any type of nucleic acid can be fragmented in a random manner. Thus, our process makes it possible to prepare detection probes, for example. Finally, the phosphate is only a linker arm between the nucleic acid and the label.
No process of fragmenting before labelling in one or two steps has been described in the prior art.
The present invention therefore proposes a process which overcomes the previously mentioned drawbacks. Thus, this process makes it possible to obtain RNA fragments which are uniformly labelled once the fragmentation has been completed. In addition, the fragmentation makes it possible to obtain fragments which are of an optimum size for a possible hybridization. With the quality of the hybridization having been improved, the detection of this hybridization will be more rapid and efficient.
Labelling is understood as being the attachment of a label which is able to generate a detectable signal either directly or indirectly. The following is a non-limiting list of these labels:
enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, xcex2-galactosidase and glucose-6-phosphate dehydrogenase,
chromophores, such as fluorescent and luminescent compounds and dyes,
groups having an electron density which can be detected by electron microscopy or by their electrical properties such as conductivity, amperometry, voltametry and impedance,
detectable groups, for example whose molecules are of sizes which are sufficient to induce detectable modifications in their physical and/or chemical characteristics; this detection can be effected by means of optical methods such as diffraction, surface plasmon resonance, surface variation and angle of contact variation, or physical methods such as atomic force spectroscopy and the tunnel effect,
radioactive molecules such as 32P, 35S or 125I.
Indirect systems can also be used, such as ligands which are able to react with an anti-ligand. Ligands/anti-ligand pairs are well known to the skilled person, as, for example, in the case of the following pairs; biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin and polynucleotide/complementary polynucleotide. In this case, it is the ligand which carries the binding agent. The anti-ligand can be detected directly by means of the labels described in the preceding paragraph or be itself detectable by means of a ligand/anti-ligand.
To this end, the present invention relates to a process for labelling a synthetic or natural ribonucleic acid (RNA), characterized in that it consists:
in fragmenting the RNA, and
in labelling at the level of the terminal phosphate which is located at the 3xe2x80x2 end and/or the 5 xe2x80x2 end of each fragment of the said RNA, the said terminal phosphate having been freed during the fragmentation.
According to a preferred mode of operation, the labelling of tile 3xe2x80x2 end of each fragment of the RNA is effected in the case of each fragment apart from the fragment which constitutes the 3xe2x80x2 and/or 5xe2x80x2 end of the starting RNA, and/or the labelling of the 5xe2x80x2 end of each fragment of the RNA is effected in the case of each fragment apart from the fragment which constitutes the 5xe2x80x2 end of the starting RNA.
According to a first embodiment, the fragmentation and the labelling are effected in one step.
According to a second embodiment, the fragmentation and the labelling are effected in two steps.
Whatever the embodiment, labelling of the 3xe2x80x2 end or the 5xe2x80x2 end of an RNA fragment is effected by binding a reactive function which is carried by a label, or which is capable of being subsequently linked to at least one label, to the phosphate which is in the 2xe2x80x2 position, in the 3xe2x80x2 position or in the cyclic monophosphate 2xe2x80x2-3xe2x80x2 position, with respect to the ribose. When there are at least two labels, the technique is then a signal-amplification technique.
The fragmentation and/or the labelling of the 3xe2x80x2 end or the 5xe2x80x2 end of an RNA fragment is effected by binding a nucleophilic, electrophilic or halide function which is carried by a label, or which is capable of being subsequently linked to a label, to the phosphate in the 2xe2x80x2 position, in the 3xe2x80x2 position or in the cyclic monophosphate 2xe2x80x2-3xe2x80x2 position, with respect to the ribose.
The fragmentation of the RNA is effected enzymically, chemically or physically.
The enzymic fragmentation of the RNA is carried out by means of nucleases.
The chemical fragmentation of the RNA is carried out by means of metal cations which may or may not be combined with a chemical catalyst.
In this case, the metal cations are Mg++, Mn++, Cu++, Co++and/or Zn++ions and the chemical catalyst consists of imidazole, a substituted analogue, for example N-methylimidazole, or any chemical molecule which has an affinity for the RNA and which carries an imidazole nucleus or a substituted analogue.
The physical fragmentation of the RNA is carried out by means of sonication or by means of radiation.
In all the cases in point, the labelling of the 3xe2x80x2 end or the 5xe2x80x2 end of an RNA fragment is effected by binding a molecule Rxe2x80x94X, where R consists of the label and X is the agent for binding the label to the RNA, such as a hydroxyl, amine, hydrazine, alkoxylamine, alkyl halide, phenylmethyl halide, iodoacetamide or maleimide grouping, to the phosphate which is linked to the 2xe2x80x2 position, to the 3xe2x80x2 position or to the cyclic monophosphate 2xe2x80x2-3xe2x80x2 position of the ribose.
The present invention also consists of an RNA fragment which is obtained by the process, in accordance with the features expounded above, which is characterized in that the RNA fragment comprises, on the one hand, a single nucleotide which is labelled at the level of the terminal phosphate which is located at the 3xe2x80x2 end or the 5xe2x80x2 end of the RNA fragment, the said terminal phosphate having been freed during the fragmentation, and, on the other hand, at least one other nucleotide whose base (purine: adenine/guanine or pyrimidine: uracyl/cytosine) is identical to that of the labelled nucleotide.
This RNA fragment comprises from 10 to 100 nucleotides, preferably from 30 to 70 nucleotides and preferably from 40 to 60 nucleotides.
According to a preferred embodiment, the RNA fragment comprises at least one thiophosphate nucleotide.
In addition, the labelled nucleotide is a thiophosphate nucleotide.
The invention relates to the use of an RNA fragment, as defined above, as a probe for detecting an RNA and/or a DNA or an RNA fragment and/or a DNA fragment.
The invention finally relates to the use of an RNA fragment, as defined above, as a labelled target which is able to bind to a capture probe.